The Orthomyxoviridae is a family of RNA viruses which infect vertebrates. The family includes those viruses which cause influenza.
Influenza is a viral infection of the respiratory system characterized by fever, cough, and severe muscle aches. There are three genera of influenza virus, identified by antigenic differences in their nucleoprotein and matrix protein: Influenzavirus A, Influenzavirus B and Influenzavirus C.
Influenza A and B viruses each contain eight segments of single stranded RNA (ssRNA). The viruses comprise major external virion proteins, haemagglutinin (HA) and neuraminidase (NA). The Influenzavirus A genus comprises 16 HA subtypes and 9 NA subtypes which probably form all 144 possible permutations. However, influenza B is a single subtype and is significantly distinct from influenza A.
Influenza C virus contains seven segments of ssRNA, because the virus lacks a separate neuraminidase gene (see Lamb, R. and Krug, R. M. (1996) Chapter 45; Orthomyxoviridae: The viruses and their replication—Fields Virology, 3rd Edition, Raven Publishers, Philadelphia).
Members of the family Paramyxoviridae are viruses with a narrow specific host range in nature, but in cultured cells they display a broad host range. Features of infection in vivo include the formation of inclusion bodies and syncytia. Paramyxovirus infection typically begins in the respiratory tract and may remain at that site (e.g., human respiratory syncytial virus, HRSV) or may spread to secondary sites (e.g., lymphoid and endothelial tissues for measles virus (MeV)). In general, paramyxovirus infections are limited by, and eliminated by, host immunity. However, virus sometimes can be shed for periods of weeks or months in normal and, especially, in immunocompromised individuals.
A genus within the Paramyxoviridae is Pneumovirus. This genus includes bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) of which there are two subtypes designated A and B and several virus strains e.g. HRSV strain A2, HRSV strain RS-S2 and BRSV strain Snook. There is also a murine pneumonia virus (also known as pneumonia virus of mice: PVM)
Antiviral drugs are a class of medication used specifically for treating viral infections. Specific antivirals are frequently used for specific viruses. Most of the antivirals now available are designed to help deal with HIV, herpesviruses (best known for causing cold sores and genital herpes but actually causing a wide range of diseases), the hepatitis B and C viruses (which can cause liver cancer), and influenza A and B viruses. Researchers are now working to extend the range of antivirals to other families of pathogens.
For example, Amantadine and Rimantadine are effective against all influenza type A viruses, but not influenza type B viruses. Viruses that are resistant to these compounds are commonly found. Other anti influenza A and B virus include Zanamivir (Relenza®) and Oseltamivir (Tamiflu®). However, the effectiveness of these therapies is somewhat limited. Treatment has to be started soon after infection and it needs to be given twice daily. Antiviral treatment is only able to shorten the duration of symptoms by one to three days. Viruses that are resistant to Tamiflu are increasingly being found in patients with influenza.
Vaccines are used to generate an immune response to attack viruses. Vaccines traditionally consist of a weakened or a killed version of the pathogen. More recently “subunit” vaccines have been devised that consist strictly of protein targets from the pathogen. Vaccines stimulate the immune system without doing serious harm to the host, and so when the real pathogen attacks the subject, the immune system responds to it quickly and blocks it.
Vaccines may be effective in combating infection by stable viruses, but are of limited use in treating a patient who, has already been, or is persistently, infected. Vaccines against rapidly mutating viruses, such as influenza, are problematic. The vaccine for influenza has to be updated each year. A vaccine against HIV, for example, remains elusive.
Defective interfering (DI) viruses have a long history. They were discovered as auto-interfering elements in influenza A virus preparations by von Magnus who studied them in the late 1940s and early 1950s (e.g. von Magnus, P (1947) Ark. Kemi. Mineral. Geol, 24b: 1). For many years these interfering elements were named after him. Later, when it was realized that these elements were found almost universally amongst viruses, they were called DI viruses (see e.g. Huang & Baltimore (1970) Nature 226: 325-327). Interest in DI viruses reached a peak in the 1970s but then waned due to an over-extravagant expectation of their in vivo antiviral activity.
All influenza A viruses have a replication apparatus that allows the exchange of genome segments (reassortment) in dually infected cells, giving these viruses immense genetic flexibility. Such an event gave rise to the 1957 and 1968 pandemic influenza viruses. In addition to the normal replication process, mistakes in replication occur that give rise to small RNAs of 400-500 nucleotides (nt) lacking around 80% of the central sequence of the template, which appears to result from the polymerase copying the initial part of the template, detaching from the template and then rejoining and copying the other terminus. These small RNAs retain the terminal replication and encapsidation signals. Their small size suggests that more copies can be made in unit time compared with the full-length RNA segment. Encapsidation of genomic RNAs appears to be an organized process so that a virion contains just one copy of each of the 8 segments. A virion does not appear to discriminate between a defective and a full-length RNA, so when defective RNAs are in excess they are encapsidated more frequently than the intact genome segment which they replace. A particle containing the deleted genome segment cannot synthesize the viral protein(s) normally encoded by that RNA, and is non-infectious, although it can be replicated in trans when that cell is infected by an influenza A virus. Incorporation of defective RNAs into virions results in a reduction in the amount of infectious virus produced. Thus virions carrying a deleted genome were known as interfering or defective-interfering (DI) viruses.
Viruses of the Orthomyxoviridae family give rise spontaneously to defective RNA segments as a result of an internal deletion (75-80% of the nucleotides) in one or more genomic segments. The DI virus genome is therefore a deleted form of the genome of the infectious virus which gave rise to it; and it has several unique properties which distinguishes it from other types of defective viral nucleic acid molecules (see Dimmock, N. J. (1996) “Antiviral activity of defective interfering influenza virus in vivo”—Viral and other infections of the human respiratory tract; S. Myint and D. Taylor-Robinson (Eds.), Chapman & Hall).
Compared to an active, i.e. live or infectious virus, a DI virus, while able to infect a cell, replicates and is propagated only when its genome is present in a cell which has been infected by a virus with a complete genome (sometimes referred to as a “helper virus”). DI influenza virus is encapsidated into virus particles which are usually indistinguishable in size and protein composition from infectious helper virus particles.
After arising, de novo, a DI genome is rapidly amplified in concentration relative to that of the genome of the infectious virus, so that within a few infectious cycles (or passages) there is more DI virus in a population than infectious helper virus.
DI virus has the ability to interfere intracellularly with infectious helper virus so that it is specifically able to inhibit multiplication of infectious helper virus.
In vivo animal studies have shown that spontaneously produced DI influenza A virus (A/equine/Newmarket/7339/79 (H3N8)) can, in sufficient amount, protect mice against lethal influenza A challenge with both the homologous virus (EQV) or with heterologous subtypes A/WSN (H1N1) or A/PR/8/34 (H1N1). In these studies the DI virus preparation was UV-treated in order to inactivate the infectivity of any live helper virus present. This amount of UV irradiation does not inactivate the interfering or protecting activity of the DI virus. A single administration appeared to provide prophylaxis for up to about 5 days. However, these DI influenza A virus preparations were heterogeneous and comprised a multiplicity of undefined defective RNA sequences from different genomic segments (see Noble and Dimmock (1994) Journal of General Virology 75: 3485-3491).
DI influenza A virus A/WSN (H1N1) grown in embryonated chicken's eggs protected mice against lethal challenge with A/WSN (H1N1). Comparison of egg-grown DI virus RNA species with DI virus RNA extracted from surviving mouse lungs showed that there were at least 5 putative DI RNA sequences present. Each of the five RNA species of the DI virus had an internal deletion (see Noble & Dimmock (1995) Virology 210: 9-19). The 3′ and 5′ ends of four of these RNA molecules appeared intact.
Duhaut & Dimmock (2000, Virology 275: 278-285) modified a defective segment 1 RNA of EQV by placing it under the control of a human RNA polymerase I promoter (Pol I) in a plasmid. Each of the plasmids encodes RNA of approx 400 nucleotides but, due to the exact position of the internal deletion, differing lengths of the 5′ and 3′ end sequences remained. Vero cells were transfected with each plasmid together with plasmids encoding the influenza virus PB1, PB2, PA, and NP proteins and the cells were then infected with one of three different helper virus subtypes, including the parent (H3N8) or an H2N2 or H1N1 subtype. Serial passage was carried out in cell culture. At least 150 nucleotides at the 5′ end of the DI virus RNA were found to be necessary for reliable passage in vitro in each of the cell lines used together with the particular helper viruses used.
It has not been possible to experimentally elucidate the process by which non-cloned DI influenza A viruses reduce the yield of infectious virus, inhibit virus-induced cytopathology, and protect animals from clinical disease, as most populations of DI influenza A virus contain many different defective RNA sequences, derived from different genome segments and with a variety of central deletions. Thus the RNA content of such non-cloned populations of defective virus cannot be reproduced effectively, and it has not been possible to analyse the relationship between RNA sequence and antiviral activity.
Duhaut & Dimmock (2002, J. Gen. Virol. 83: 403-411) demonstrated that a DI influenza A virus RNA derived from a plasmid system appears to behave authentically in cell culture. One plasmid (POLI-317) gave rise to DI virus RNA that replicated stably in vitro in the presence of helper virus and strongly inhibited the production of the helper virus in that system.
Duhaut & Dimmock (2003, Journal of Virological Methods 108: 75-82) described the preparation of a defined (i.e. molecularly cloned) DI influenza A virus generated entirely from plasmids which were used to transfect host cells in culture. The plasmids used encoded the DI RNA (H3N8 or H7N7) and infectious influenza virus (A/WSN, H1N1). DI influenza A virus generated in this way was passaged once in embryonated chicken's eggs and then administered to mice in the presence of helper virus (H1N1). The cloned DI virus propagated intact within mouse lung. The cloned DI virus (without infectious helper) was also tested for any protective effect in mice against a lethal (H1N1) challenge. Some very weak and short lived prophylactic effect was observed, but this only delayed the onset of clinical symptoms and death in the mice.
Noble et al. (2004, Vaccine 22: 3018-3025) reported an in vivo study in mice using a naturally occurring (i.e. heterogeneous and undefined) DI influenza A virus preparation (EQV H3N8). Administration of this DI virus preparation to mice was found to generate prophylaxis protection for a period, and at the same time converted an otherwise lethal infection into an avirulent and immunizing infection.
Dimmock & Marriott (2006, Journal of General Virology 87: 1259-1265) described an apparent anomaly in which a heterogeneous and undefined DI influenza A virus preparation solidly protects mice from lethal disease caused by A/PR/8/34 (H1N1) and A/WSN/40 (H1N1) viruses, but only marginally protects from disease caused by A/Japan/305/57 (A/Jap H2H2). A/Jap was found to require 300-fold more infectious units to cause clinical disease in mice than A/PR8. The proportions of DI virus and challenge virus were varied and tested. A conclusion reached was that the efficacy of the DI virus depends on the infectious dose of challenge virus rather than its disease-causing dose.
Mann et al. (2006, Vaccine 24, 4290-4296) tested heterogeneous and undefined DI A/EQV RNAs that had been rescued by A/PR8 in ferrets. DI influenza A virus was administered in two doses followed by challenge with infectious A/Sydney 5/97 (H3N2). The DI virus-treated ferrets showed only occasional and mild clinical symptoms, compared to the control animals which became severely ill.
US2006/0057116 A1 (Kawaoka and Neumann) describes plasmids and a method of transfecting and culturing cells to produce recombinant influenza A virus in vitro in the absence of any helper virus. Specifically, influenza A viruses can be prepared entirely from their cloned cDNAs in transfected cell lines. Mutations can be incorporated into any gene segment.
WO2006/051069 (Solvay Pharmaceuticals and Erasmus University) discloses conditionally defective influenza virus particles and a method of making them. From the starting point of transfected cells not being able to produce large quantities of defective influenza virus particles for use as vaccines, the specification teaches an alternative method. The method involves a cell transfected with plasmids encoding seven RNA segments of the influenza virus but missing an eighth segment that expresses a polymerase protein. The cell includes a second expression plasmid carrying the sequence of the missing polymerase gene. On expression, the transfected cell yields “conditionally” defective virus particles which can only replicate in a cell line expressing the polymerase protein gene that is not present in the defective genome. The defective influenza virus particles can only replicate once in suitable, albeit not complemented, host animals or cells. The conditionally defective virus particles are intended for vaccine use or gene delivery purposes and so advantageously the virus particle preparations are unable to replicate in normal cells and contain no wild-type or helper virus.
Although a prototype system has been described (see Duhaut & Dimmock, 2003 supra) for preparing a cloned DI influenza A virus (which turned out to be only weakly protective on one occasion in mice), it does not offer a practical route for preparing the necessary amounts of cloned DI viruses needed for further laboratory investigations, let alone the amount of cloned DI virus that would be needed on a routine basis in order to carry out animal and human clinical trials or provide for prophylaxis and/or therapy in routine, epidemic or pandemic situations.
Huang, A. S. & Baltimore, D. (1970) “Defective viral particles and viral disease processes” Nature (Lond) 226, 325-327. This review article at page 325 describes how the synthesis of DI particles by cells or animal tissues on infection with high multiplicities (or undiluted passage virus) is achieved for Rift Valley fever virus, vesicular stomatitis virus, fowl plague virus, simian virus 40, polyoma virus, lymphocytic choriomeningitis virus, Sendai virus, simian virus 5, and poliovirus.
Holland, J. J. (1990) “Defective viral genomes” In Virology, 2nd edn. pp. 151-165. Edited by B. N. Fields & D. M. Knipe New York: Raven Press. In this review article, page 155 describes how serial undiluted passage of virus in cell culture (or eggs or animals) is still the method of choice for generation of DI particles of any virus.
Nayak, D. P., Chambers, T. M. & Akkina, R. K. (1985) “Defective-interfering (DI) RNAs of influenza viruses: origin, structure, expression and interference” Current Topics in Microbiology and Immunology 114, 103-151 is a review article which attests to the production of DI viruses by serial independent undiluted passage of virus.
WO2007/135420 (University of Warwick) describes a method of producing previously unavailable and sufficient quantities of cloned DI influenza A virus for experimental or clinical use in preventing or treating influenza A infection in humans or animals, including birds. Also described is the medical use of cloned DI influenza A as a prophylactic or therapeutic treatment of influenza A infection. The cloned DI influenza A virus is identified as having an antiviral effect on the same or different strains of infectious influenza A.
There is a continuing need to provide antiviral treatments against the range of virus infections in humans and animals, particularly against viruses where there are no existing antiviral treatments available, against viruses that exhibit rapid mutation or against viruses which may become resistant to existing antiviral medicaments and against newly discovered or poorly characterised viruses.
The inventors have made an unexpected discovery which is that cloned human DI influenza A virus protects against infection by a different (heterologous) virus. More particularly, the inventors have found that human DI influenza A virus protects mice subjected to a lethal challenge of pneumonia virus of mice (PVM). The inventors have also surprisingly discovered that cloned DI influenza A virus is capable of protecting mice against lethal challenge with infectious influenza B virus. This is an unexpected finding because, although influenza A and B viruses belong to separate genera within the same family of viruses, they do not interact genetically.
The inventors made a further, unexpected discovery which is that cloned DI influenza A virus administered to mice results in a stimulation of natural interferon production. Without wishing to be bound by any particular theory, the inventors believe that a mechanism by which cloned DI viruses may exert their antiviral effect against heterotypic viruses is by stimulating the localised production of natural interferon at the site of cloned DI virus administration.
Accordingly, in one aspect the present invention provides cloned, defective interfering (DI) virus for use in the prevention or treatment of virus infection or disease in an individual, wherein the infection or disease is caused by a virus which is different to the virus from which the DI virus is derived.
Cloned DI viruses included those which have single stranded RNA, as well as those which are double stranded RNA, single stranded DNA or double stranded DNA.
In another aspect the invention provides cloned, defective interfering (DI) influenza A virus for the prevention or treatment of an infection or disease caused by a virus other than influenza A.
A virus different to the virus from which the DI virus is derived is preferably a wild type strain of another virus. For example if the DI virus is DI influenza A virus, then the different virus is human respiratory syncytial virus (HRSV).
Cloned DI virus materials for use in the invention are preferably substantially uniform in terms of DNA or RNA nucleic acid sequence. The uniformity may be more than a percentage in the range 95-99.9% and 100% genetic uniformity, as measured by sequence % identity, may be provided.
Preventative (prophylactic) and therapeutic treatments (i.e. post infection when clinical symptoms are manifest) are possible within the scope of the invention. Therapeutic treatment may be administered when a suspected viral infection is still sub-clinical.
The infection or disease to be prevented or treated in accordance with the invention may be one caused by a respiratory or a systemic virus.
Such infection or disease may be caused by a virus of the Paramyxoviridae, such as a Pneumovirus, e.g. human respiratory syncytial virus (HRSV).
The infection or disease may be caused by a Metapneumovirus, e.g. human metapneumovirus (HMPV).
In other aspects of the invention the infection or disease may be caused by a virus of the Orthomyxoviridae, such as influenza B virus or influenza C virus.
In further aspects of the invention, the infection or disease may be caused by a hepatitis virus or a virus of the Hepadnaviridae, e.g., hepatitis B virus (HBV).
The infection or disease may be caused by a virus of the Flaviviridae, e.g. Hepacivirus. The virus may be hepatitis C virus (HCV).
In yet further aspects of the invention, the infection or disease may be one caused by Papillomaviridae, e.g. papillomavirus.
The virus causing the infection or disease can be one infectious against humans and other animals (including birds, reptiles or amphibians).
In accordance with the invention, cloned DI virus may be administered via the nose and/or via the lungs of an individual human or animal. Alternatively, the cloned DI virus may be administered parenterally, e.g. subcutaneously, intramuscularly, intravenously or intraperitoneally.
The invention also provides a method of stimulating innate immunity and natural interferon production by a cell, tissue, organ, or in a human or in an animal, comprising administering an interferon-inducing amount of a defective interfering (DI) virus to the cell, tissue, organ, human or animal, provided that the DI virus is not an RNA virus with a “copyback” or “snapback” RNA structure when DI virus is administered to a cell in cell culture.
In such methods of the invention, the DI interfering virus is preferably other than a DI vesicular stomatitis virus or DI Sendai virus when DI virus is administered to a cell in cell culture.
The stimulation of natural interferon production is readily measured by the person of skill in the art using known assays. The interferon produced may be of any of the naturally inducible type I interferons; namely interferon (IFN) type I and mainly IFN-α and IFN-β (Theofilopoulos, A. N., Baccala, R., Beutler, B. and Kono, D. H. 2005 Type I interferons (α/β) in immunity and autoimmunity. Annual Review of Immunology 23, 307-336).
In stimulating innate immunity and natural interferon production in accordance with the invention, the DI virus is preferably a cloned DI virus. More particularly the stimulation of natural interferon may employ a host replication competent helper virus. In further such embodiments the cloned DI virus may be a different strain of virus or may be heterotypic in relation to the helper virus.
When stimulating interferon production, then administration of DI virus needs to take place in advance of the desired antiviral effect. This is because there is a lag between administration and the reaction of the body whereby cells generate interferon. Normally a lag of 24 hours is seen and so administration is preferably about 24 hours or more prior to the desired level of interferon production.
The invention therefore also includes a method of preventing or treating a viral infection in an individual human or animal comprising administering an interferon-inducing amount of a defective interfering (DI) virus to the individual.
In such aspects of the invention, DI virus is preferably administered via the nose and/or via the lungs of the individual, although parenteral, e.g. subcutaneous, intramuscular, intravenous or intraperitoneal administration is possible.
In accordance with the aforementioned methods of the invention, DI virus may not be given directly to an individual, but additionally or alternatively, cells, tissue or organ may be isolated from the human or animal body, i.e. ex vivo and/or in vitro, treated with the DI virus in order to induce interferon production, and then reintroduced or transplanted back into the body of the same or another receptive individual or the same or similar blood or tissue type.
In accordance with all aspects of the invention defined above, the DI virus may be a DI influenza virus, preferably a DI influenza A virus, or a DI Semliki Forest virus.
The inventors therefore provide antivirals and antiviral treatments based on defective interfering viruses that have the capability of protecting against heterotypic (i.e. different) virus infection in any host. For example, in treating pulmonary viral infections, the antiviral medicament is preferably delivered by intranasal administration to the cells of the respiratory tract. Other routes of administration may include mucosal, pulmonary and oral cavity. Other routes include gastro-intestinal via oral administration.
An advantage of cloned DI virus is that an individual known or suspected of being infected with any virus can be treated for the infection, even if symptoms of infection have yet to be observed or infection diagnosed. The individual can be administered with the cloned DI virus medicament as soon as possible when an infection is suspected. Individuals can also be treated as soon as possible after having been in contact with other individuals of the same or different species and who are known or suspected to be infected with the virus. Advantageously, protection is not believed to involve an adaptive immune response and is achieved on administration of the cloned DI virus medicament alone without the need for administration of infectious helper virus. There is therefore no requirement to administer the cloned DI virus in advance of infection like a conventional vaccine (which relies on B cell and T cell-directed immune responses in order to generate a protective effect), although 24 hours pretreatment with DI virus is advantageous.
Medicaments in accordance with the invention may be administered to individuals on a precautionary basis.
The individual to which the medicament may be administered may be an animal or human, preferably wherein the animal is selected from a pig, horse, dog, cat or bird (wild or domesticated). In the case of birds, whether wild or domestic, the medicament may be administered conveniently via the oral tract, e.g. by incorporating the medicament in drinking water or in food. In the case of bird species, preferred domesticated species include, for example, duck, goose, turkey, or hen e.g. broiler chicken.
A dosage regime in accordance with the invention may consist of a single dose of medicament. The amount of cloned DI virus in a medicament can be measured by quantitative RT-PCR. Probes and/or primers specific for the deleted RNA or DNA segment are employed.
The amount of cloned DI virus in a medicament may be of the order (per dose) of 1 ng-10 μg of virus (measured in terms of total virus protein). The amount of DI virus may be in the range 0.05 μg-10 μg. Preferred embodiments include 0.01-0.1 μg, 0.01-1 μg or 0.01-10 μg of virus protein, more preferably 10 ng, 100 ng, 1 μg or 10 μg virus protein.
Where cloned DI influenza A virus is used as the basis of a medicament in accordance with the invention, the amount of cloned DI virus in the medicament may be in the range per dose of 0.05-5000 HAU, preferably a range selected from 0.1-100, 0.5-50 or 1-10 HAU. Other possible ranges include 0.05-10 HAU, 0.1-50 HAU, 1-100 HAU and 1-5000 HAU.
The amount of cloned DI virus, whether measured in terms of HAU or μg virus protein per dose, may be varied according to the subject. For example, a horse may require 4× the human dose, whereas a bird may require 1/10 of the human dose.
The defective nucleic acid in cloned DI virus may have at least one deletion compared with the genomic segment from which it derived, although a multiplicity of deleted portions of segment 1 may occur. The deletions may be separated by a multiplicity of contiguous nucleotides. The sequence of the defective nucleic acid may contain one or more nucleotide changes compared with the genomic segment from which it derived. These may comprise a different replacement nucleotide, a deleted nucleotide or an inserted additional nucleotide.
The 5′ and 3′ ends of the genomic segment including the deletion are preferably intact. In a more preferred embodiment the segment is segment 1. The effect of deletion is that the 5′ end of the segment of virion RNA has at least 150, 200 or 220 nucleotides. Preferably the 5′ end of the segment has a number of nucleotides in the range 150-500, more preferably 150-250, or 150-220.
In terms of the 3′ end, the remaining (undeleted) portions comprise at least 20, 50, 100, 200, 300, 400 or 500 nucleotides. The 3′ end of segment 1 may have a number of (undeleted) nucleotides in the range 20-600, 30-550, 40-500, 50-450, 60-400 or 75-250.
The segment deletion may be at least 50% of the nucleotides, preferably at least 75%, more preferably at least 80% of the nucleotides. In order for an effective deletion in the RNA segment, at least one nucleotide may be deleted. In more preferred embodiments, the deletions may consist of at least 3, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 750, 1000, 1500, 3000 or 5000 nucleotides, preferably contiguous nucleotides. A multiplicity of deletions is possible within the same RNA segment.
The invention therefore provides a pharmaceutical composition comprising a cloned DI virus as hereinbefore described.
Pharmaceutical compositions of the present invention, suitable for administration, comprise the DI virus, in sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions may further comprise auxiliary agents or excipients, as known in the art, see, e.g., Berkow et al., The Merck Manual, 16th edition Merck & Co., Rahman, N.J. (1992), Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore, Md. (1987) & Osol (ed.), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1324-1341 (1980). The composition of the invention is preferably presented in the form of individual doses (unit doses).
Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, e.g. purified water. As well as inert diluents, exemplary compositions may also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavouring, or perfuming agents.
When a composition or medicament of the present invention is used for administration to an individual, it may further comprise salts, buffers, or other substances which are desirable for improving the efficacy of the composition.
Preferred compositions or medicaments are for mucosal delivery. Of the various mucosal delivery options available, the intranasal route is the most practical as it offers easy access with relatively simple devices that have already been mass produced. The composition of the invention is thus preferably adapted for and/or packaged for intranasal administration, such as by nasal spray, nasal drops, gel or powder (see Almeida & Alpar (1996) J. Drug Targeting 3: 455-467 and Agarwal & Mishra (1999) Indian J. Exp. Biol. 37: 6-16.).
Other possible routes for mucosal delivery include oral, intragastric, pulmonary and intestinal. The composition of the invention may be adapted for and/or packaged for mucosal administration (e.g. see Walker (1994) Vaccine 12: 387-400, Clements (1997) Nature Biotech. 15: 622-623 & McGhee et al. (1992) Vaccine 10: 75-88). For oral administration tablets or capsules (optionally enteric-coated), may be provided.
Optionally, liquid, transgenic plant material, drops, inhaler, aerosol, enteric coating, suppository, pessary, etc. (see Michetti (1998) J. Gastroenterol. [Suppl X]: 66-68 and chapter 17 of Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995).
The composition of the invention may be administered by a parenteral route. The parenteral route includes intravenous, intramuscular, subcutaneous, intradermal, epicutantous/transdermal, or intraperitoneal administration. Various types of parenteral formulations may be used, including solutions, micellar dispersions, emulsions, inclusion complexes (cyclodextrins), liposomes and suspensions.
Whatever route of delivery is used, compositions or medicaments of the invention are preferably in unit dose form. Effective doses can be routinely established. For example, a typical human dose of the composition for injection or for intranasal use has a volume between 0.1-0.5 ml e.g. two 100 μl sprays, one per nostril.
Compositions of the invention are preferably sterile and preferably not pyrogenic. At higher concentrations, a DI influenza A virus composition may be pyrogenic or exhibit residual pyrogenic activity. The compositions are preferably buffered, e.g. at between pH 6.5 and pH 8, generally around pH 7.
An advantageous form of nasal administration is described in WO2006/041819 (Medimmune).
The invention also includes a method of converting a virulent virus infecting a subject into an avirulent virus infection, comprising administering to the subject an effective amount of a cloned DI virus.
The cloned DI virus particle may be administered before, simultaneously or after the infection.
The invention includes a method of converting a virulent virus infecting a subject into an avirulent virus infection that vaccinates the subject against the infecting virus, comprising administering to the subject a cloned DI influenza A virus.
The subject may, or may be suspected of being, infected with a given virus. In cases of actual or even suspected infection, cloned DI virus may be administered as soon as possible, within 48 hours, preferably within 24 hours of the individual being infected, or being suspected of being infected. Similarly, individuals can be administered the cloned DI virus as a precautionary measure if they are shortly to be exposed to infectious virus, whether from infected humans, animals or birds. Persons having to deal with animal or bird carcasses or with human corpses known or suspected of being infected with pathogenic viruses, particularly in epidemic or pandemic situations can be administered the cloned DI virus of the invention. Such persons can be administered the medicament of the invention on a precautionary basis immediately prior to risk of virus exposure. Unlike the employment of a vaccine, it is not necessary to know the identity of the infecting virus.
In each of the methods of the invention described above, the cloned DI virus is administered in sufficient amount. The helper virus may be of any corresponding strain of the virus, whether from humans or other animals, including birds.
Cloned DI virus also protects when given up to 24 hours after infection and beyond. It is thus able to counter an actual infection. It can therefore also be used as a treatment for family and other direct contacts of infected individuals.
Cloned DI virus is easy to administer. A drop of saline containing the cloned DI virus can simply be squirted up the nose. Aerosol administration, used already for some vaccines, offers another simple route of administration. The cloned DI virus provides a useful treatment for domestic animals, e.g. via drinking water.
The inventors have carried out experiments and unexpectedly found that some influenza A DI viruses afford mice a degree of protection from influenza B infections. The order of efficacy of DI viruses tested against influenza B was found to be:244/PR8=220/Vic>220/PR8i.e. 244/PR8 and 220/Vic and are more efficacious than 220/PR8.
Whereas the order of efficacy of DI viruses against influenza A (see Tables 1 and 2 below) is:244/PR8>220Nic=317Nic>220/PR8i.e. 244/PR8 and 220/Vic are the most efficacious against both influenza A and influenza B viruses and 220/PR8 is the least efficacious. Tests with 317/Vic are not complete.
However, when DI influenza A virus was administered 24 hours before infection with influenza B virus the order of efficacy of DI viruses against influenza B was found to be:244/PR8>220/PR8i.e. Under these conditions 244/PR8 is again the most efficacious and 220/PR8 the least efficacious.
TABLE 1Summary of protection by 244/PR8 and 244/WSN DI influenza A viruses against A/WSN: the lowest effective dose is highlighted793, 797, 804, 812 and 813 are all independent but equivalent preparations of 244/PR8.*New titration of WSN: 640x;**New dilution and titration of WSN: 640a.Nd, not done.
TABLE 2Summary of protection by 220/Vic, 220/PR8 and 317/Vic DI influenza A viruses against A/WSN: the lowest effective dose is highlightedNd, not done.
Protection from influenza B was found to require 100-fold more DI influenza A than required to protect against influenza A infection (see Table 6). Influenza B has a similar ID50:LD50 to the influenza A viruses used, and so the low DI influenza A efficacy does not appear due to an excessively high dose of influenza B challenge virus.
It is unlikely that influenza A DI RNAs swamp the influenza B infection by the more rapid replication of the small DI RNA as influenza and B viruses are not known to interact genetically. For example, all attempts to make influenza A×influenza B hybrid viruses (reassortants) have failed. In addition the inventors have found that influenza B does not support the replication of influenza A DI RNA 244.
Active DI viruses have also been found to protect mice from B/Lee by the stimulation of alpha/beta interferons in the respiratory tract and the inventors have found that:                (a) DI influenza A protects mice from infection with the heterologous pneumonia virus of mice (PVM) in the mouse.        (b) Intranasal DI influenza A (with no infectious helper virus and no challenge virus) stimulates interferon in the lungs of mice, as demonstrated by in vitro antiviral bioassay.        
A 1/100th of the dose of DI influenza A that protects mice from B/Lee also protects mice from influenza A. This suggests that interferon alpha/beta is not essential to protection from influenza A—this is corroborated by a finding that mutant mice that lack interferon alpha/beta activity (because they lack the interferon alpha/beta receptor) are protected from influenza A by DI influenza A.
In protecting mice against PVM, mice are inoculated with DI influenza A virus 24 h before the virus challenge. DI influenza A virus was equally effective against influenza B infection when inoculated 24 h before the virus challenge. However, when DI influenza A virus and influenza B virus were inoculated simultaneously, a 10-fold higher concentration of DI influenza A virus was required to effect the same degree of protection.
A few other DI viruses (vesicular stomatitis virus, a rhabdovirus and Sendai virus, a paramyxovirus) are known to stimulate interferon alpha/beta in vitro. However, these are all “snapback” RNAs—i.e. single stranded RNAs whose 3′ and 5′ halves are complementary (see Marcus, P. I. & Sekellick, M. J. (1977) Nature (Lond) 266, 815-819; Sekellick, M. J. & Marcus, P. I. (1982) Virol 117, 280-285; Marcus, P. I. & Gaccione, C. (1989) Virol 171, 630-633; Strahle, L., Garcin, D. & Kolakofsky, D. (2006) Virol 351, 101-111.
A range of other DI viruses other than influenza A DI viruses can be used to induce interferon in animals and cells. Preferably such DI viruses are cloned and are not “snapback” or “copyback”. For example, there are various cloned DI viruses which belong to the Alphavirus genus of the family Togaviridae, and specifically to Sindbis virus and Semliki Forest virus (SFV). They are all similar in structure but differ in sequence. The SFV DI viruses have been tested for their ability to protect animals. The SFV DI viruses DI-6 and DI-19 have both been cloned [see Thomson, M. and N. J. Dimmock, Common sequence elements in structurally unrelated genomes of defective interfering Semliki Forest virus. Virology, 1994. 199: p. 354-365; Thomson, M., C. L. White, and N. J. Dimmock, The genomic sequence of defective interfering Semliki Forest virus (SFV) determines its ability to be replicated in mouse brain and to protect against a lethal SFV infection in vivo. Virology, 1998. 241: p. 215-223.] SFV has a single molecule of single stranded, positive sense RNA that comprises 11422 nt. The Alphaviruses are viruses of vertebrates that are insect-transmitted. They replicate in both the invertebrate and vertebrate hosts. Alphavirus DI viruses have been reviewed [see Kaariainen, L. and H. Soderlund, Structure and replication of alphaviruses. Current Topics in Microbiology and Immunology, 1978. 82: p. 15-69; Stollar, V., Defective interfering alphaviruses, in The Togaviruses, R. W. Schlesinger, Editor. 1980, Academic Press: New York. p. 427-457; Schlesinger, S. and B. G. Weiss, Defective RNAs of alphaviruses, in The Togaviridae and Flaviviridae, S. Schlesinger and M. J. Schlesinger, Editors. 1986, Plenum Press: New York and London. p. 149-169; Barrett, A. D. T. and N. J. Dimmock, Defective interfering viruses and infections of animals. Current Topics in Microbiology and Immunology, 1986. 128: p. 55-84; Dimmock, N. J., The biological significance of defective interfering viruses. Reviews in Medical Virology, 1991. 1: p. 165-176.3-7]. SFV DI-19 is 1244 nt long and has 10.9% of the virion genome. SFV DI-19 has no major double stranded region. In terms of biological activity, the DI-19 virus interferes in cell culture with SFV multiplication and RNA replication and protects mice from SFV-mediated disease. SFV is neurotropic and when mice are inoculated intranasally, the virus enters the brain probably by entering and ascending the olfactory nerve, and causes encephalitis and usually fatal disease. Co-administration of SFV DI-19 with SFV protects against such disease.
Many viruses give rise to defective genomes and many of such defective viruses will be interfering viruses. The following are given as a list of non-limiting examples of viruses where defective genomes may be found:
Family Herpesviridae
                Herpes simplex virus        Equine herpesvirus        Pseudorabies        Cytomegalovirus        Herpesvirus        Epstein-Barr virusFamily Papovaviridae        SV40        Papovaviruses        Polyoma virusFamily Baculoviridae        BaculovirusFamily Hepadnaviridae        Hepatitis B virus        Duck hepatitis B virusFamily Adenoviridae        Adenovirus type 12        Mouse adenovirusFamily Parvoviridae        Parvoviruses        
In the examples below mice were used under the Home Office Project Licences PPL 40/2561 and PPL 40/2129 and Personal Licences 30/1253 and PIL 40/03497.